Preparation of regenerative tissue scaffolds

ABSTRACT

Devices and methods for treating or repairing a tissue or organ defect or injury are provided. The devices can include tissue scaffolds produced from acellular tissue matrices and polymers, which have a stable three-dimensional shape and elicit a limited immunologic or inflammatory response.

This application claims priority to U.S. Provisional Patent ApplicationNo. 61/317,443, which was filed on Mar. 25, 2010.

The present disclosure relates to devices and methods for treatingtissue or organ defects or injuries, including tissue scaffolds fortreating tissue defects.

Human, animal, and synthetic materials are currently used in medical andsurgical procedures to augment tissue or repair or correct tissuedefects. For certain purposes, materials with stable preformed shapesare needed. For example, for certain bone defects and soft tissuedefects, stable three-dimensional structured devices are required tocorrespond with the defect site and allow regeneration of tissue with adesired structure. However, various devices and methods for repairing orcorrecting tissue or organ defects have had certain disadvantages.

Accordingly, there is a need for improved devices with better stabilityfor medical applications.

In certain embodiments, a method for making a tissue scaffold isprovided. The method comprises dissolving a polymer in a solvent to makea solution; mixing the solution with a particulate acellular tissuematrix (ATM) to create a mixture; placing the mixture in a mold; dryingthe mixture to form a tissue scaffold with a stable three-dimensionalshape, wherein the tissue scaffold has a reduced immunological orinflammatory response when implanted in a human than the polymer alone.

In certain embodiments, a tissue scaffold is provided. The tissuescaffold comprises a particulate ATM and a polymer, wherein the ATM isencased in the polymer to form a stable three-dimensional tissuescaffold for tissue regeneration, and wherein the tissue scaffold has areduced immunological or inflammatory response when implanted in a humanthan the polymer alone

In certain embodiments, a method of treating a tissue defect isprovided. The method comprises selecting a tissue scaffold having astable three-dimensional shape, the scaffold comprising a particulateATM; and a polymer, wherein the ATM is encased in the polymer to form astable three-dimensional tissue scaffold for tissue regeneration;identifying a defect in a tissue or organ; and implanting the tissuescaffold in the defect.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 illustrates implantation of a tissue scaffold in a defect,according to certain embodiments.

FIG. 2 is a flow chart showing a process for producing a tissuescaffold, according to certain embodiments.

FIG. 3 is a graph of differential scanning calorimetry (DSC) data ofporcine acellular dermal matrix (pADM) treated with organic solvents,according to Example 2.

FIG. 4 is a graph of DSC data of pADM in presence of polymers, accordingto Example 2.

FIG. 5A is a hematoxylin and eosin stained four week sub-dermal explantcomprising poly-4-hydroxybutyrate (P4HB) under 100× magnification,according to the process described in Example 2.

FIG. 5B is a hematoxylin and eosin stained four week sub-dermal explantcomprising pADM and P4HB under 100× magnification, according to theprocess described in Example 2.

FIG. 5C is a hematoxylin and eosin stained four week sub-dermal explantcomprising P4HB under 400× magnification, according to the processdescribed in Example 2.

FIG. 5D is a hematoxylin and eosin stained four week sub-dermal explantcomprising pADM and P4HB under 400× magnification, according to theprocess described in Example 2.

FIG. 6A is a hematoxylin and eosin stained twelve week sub-dermalexplant comprising P4HB under 100× magnification, according to theprocess described in Example 2.

FIG. 6B is a hematoxylin and eosin stained twelve week sub-dermalexplant comprising pADM and P4HB under 100× magnification, according tothe process described in Example 2.

FIG. 6C is a hematoxylin and eosin stained twelve week sub-dermalexplant comprising P4HB under 400× magnification, according to theprocess described in Example 2.

FIG. 6D is a hematoxylin and eosin stained twelve week sub-dermalexplant comprising pADM and P4HB under 400× magnification, according tothe process described in Example 2.

FIG. 7A is a hematoxylin and eosin stained four week sub-dermal explantcomprising polycaprolactone (PCL) under 100× magnification, according tothe process described in Example 2.

FIG. 7B is a hematoxylin and eosin stained four week sub-dermal explantcomprising pADM and PCL under 100× magnification, according to theprocess described in Example 2.

FIG. 7C is a hematoxylin and eosin stained four week sub-dermal explantcomprising PCL under 400× magnification, according to the processdescribed in Example 2.

FIG. 7D is a hematoxylin and eosin stained four week sub-dermal explantcomprising pADM and PCL under 400× magnification, according to theprocess described in Example 2.

FIG. 8A is a hematoxylin and eosin stained twelve week sub-dermalexplant comprising PCL under 100× magnification, according to theprocess described in Example 2.

FIG. 8B is a hematoxylin and eosin stained twelve week sub-dermalexplant comprising pADM and PCL under 100× magnification, according tothe process described in Example 2.

FIG. 8C is a hematoxylin and eosin stained twelve week sub-dermalexplant comprising PCL under 400× magnification, according to theprocess described in Example 2.

FIG. 8D is a hematoxylin and eosin stained twelve week sub-dermalexplant comprising pADM and PCL under 400× magnification, according tothe process described in Example 2.

FIG. 9A is a hematoxylin and eosin stained four week sub-dermal explantcomprising hyaluronic acid benzyl ester (BHA) under 100× magnification,according to the process described in Example 2.

FIG. 9B is a hematoxylin and eosin stained four week sub-dermal explantcomprising pADM and BHA under 100× magnification, according to theprocess described in Example 2.

FIG. 9C is a hematoxylin and eosin stained four week sub-dermal explantcomprising BHA under 400× magnification, according to the processdescribed in Example 2.

FIG. 9D is a hematoxylin and eosin stained four week sub-dermal explantcomprising pADM and BHA under 400× magnification, according to theprocess described in Example 2.

FIG. 10A is a hematoxylin and eosin stained twelve week sub-dermalexplant comprising BHA under 100× magnification, according to theprocess described in Example 2.

FIG. 10B is a hematoxylin and eosin stained twelve week sub-dermalexplant comprising pADM and BHA explant under 100× magnification,according to the process described in Example 2.

FIG. 10C is a hematoxylin and eosin stained twelve week sub-dermalexplant comprising BHA under 400× magnification, according to theprocess described in Example 2.

FIG. 10D is a hematoxylin and eosin stained twelve week sub-dermalexplant comprising pADM and BHA under 400× magnification, according tothe process described in Example 2.

FIG. 11A is a hematoxylin and eosin stained four week explant comprisingchitosan and pADM under 100× magnification, according to the processdescribed in Example 2.

FIG. 11B is a hematoxylin and eosin stained eight week explantcomprising chitosan and pADM under 100× magnification, according to theprocess described in Example 2.

FIG. 11C is a hematoxylin and eosin stained four week explant comprisingchitosan and pADM under 400× magnification, according to the processdescribed in Example 2.

FIG. 11D is a hematoxylin and eosin stained eight week explantcomprising chitosan and pADM under 400× magnification, according to theprocess described in Example 2.

DESCRIPTION OF EXEMPLARY EMBODIMENTS

In this application, the use of the singular includes the plural unlessspecifically stated otherwise. In this application, the use of “or”means “and/or” unless stated otherwise. Furthermore, the use of the term“including”, as well as other forms, such as “includes” and “included”,is not limiting.

The section headings used herein are for organizational purposes onlyand are not to be construed as limiting the subject matter described.All documents, or portions of documents, cited in this application,including but not limited to patents, patent applications, articles,books, and treatises, are hereby expressly incorporated by reference intheir entirety for any purpose.

The term “acellular tissue matrix,” as used herein, refers generally toany tissue matrix that is substantially free of cells and/or cellularcomponents. Skin, parts of skin (e.g., dermis), and other tissues suchas blood vessels, heart valves, fascia, cartilage, bone, and nerveconnective tissue may be used to create acellular matrices within thescope of the present disclosure. Acellular tissue matrices can be testedor evaluated to determine if they are substantially free of cell and/orcellular components in a number of ways. For example, processed tissuescan be inspected with light microscopy to determine if cells (live ordead) and/or cellular components remain. In addition, certain assays canbe used to identify the presence of cells or cellular components. Forexample, DNA or other nucleic acid assays can be used to quantifyremaining nuclear materials within the tissue matrices. Generally, theabsence of remaining DNA or other nucleic acids will be indicative ofcomplete decellularization (i.e., removal of cells and/or cellularcomponents). Finally, other assays that identify cell-specificcomponents (e.g., surface antigens) can be used to determine if thetissue matrices are acellular.

The present disclosure provides three-dimensional tissue scaffolds totreat defects in tissues or organs. The tissue scaffolds can include anATM that has the biologic ability to support tissue regeneration. Insome embodiments, tissue scaffolds can support cell ingrowth anddifferentiation. For example, the scaffolds can be used for tissueingrowth, orthopedic surgery, periodontal applications, tissueremodeling, tissue restoration, plastic surgery, cosmetic surgery, andreplacement of lost tissue, for example to lumpectomy, parotidectomy, orexcision of tumors, as described further below.

In addition, the tissue scaffolds can include one or more polymericmaterials, as described further below. In certain embodiments, thetissue scaffolds of the present disclosure can increase the acceptanceof the polymeric component via attenuation or reduction of immunologicalor inflammatory response. As used herein, the polymeric components caninclude synthetic polymers and/or naturally occurring polymers. Incertain embodiments, the polymeric materials in the tissue scaffolds canprovide a stable three-dimensional structure to the ATM, which increasesimplant integration and biocompatibility. As used herein, the stablethree-dimensional structure will be understood to refer to a materialthat tends to maintain a predetermined shape (e.g. formed to conform toan implant site) when in a resting state.

In various embodiments, tissue scaffolds of the present disclosure canbe used for treatment at numerous different anatomical sites. Accordingto various embodiments, tissue scaffolds can be used in a wide array ofapplications. Certain exemplary applications include, but are notlimited to, absorptive dressing, dermal regeneration (for example, fortreatments of all types of ulcers and burns), nerve regeneration,cartilage regeneration, connective tissue regeneration or repair (forexample, tendon/ligament sleeve), bone regeneration, periodontalapplications, wound/foam lining, integrated bandage dressing,substrate/base for skin grafts, vascular regeneration, cosmetic surgery,metal and/or polymer implant coating (for example, to increase implantintegration and biocompatibility), and replacement of lost tissue (e.g.,after trauma, breast reduction, mastectomy, lumpectomy, parotidectomy,or excision of tumors).

In some embodiments, the tissue scaffold elicits a reduced immunologicalor inflammatory response when implanted in a human compared to than thepolymer or polymers used to produce the scaffold alone. In someembodiments, the effect of the tissue scaffold in the host can be testedusing a number of methods. In some embodiments, the effect of the tissuescaffold in the host can be tested by measuring immunological orinflammatory response to the implanted scaffold. In some embodiments,the immunological or inflammatory response to the tissue scaffold can bemeasured by a number of methods. In some embodiments, the immunologicalor inflammatory response can be measured using histological methods. Forexample, explanted scaffold can be stained, and observed undermicroscope for histological evaluation, as described further below. Insome embodiments, the immunological or inflammatory response to thescaffold can be demonstrated by measuring the number of inflammatorycells (e.g., leukocytes). In some embodiments, the attenuatedimmunological or inflammatory response to the scaffold can bedemonstrated by a reduced number of inflammatory cells, as describedfurther below. For example, inflammatory cells can be measured throughimmuno-histochemical staining methods designed to identify lymphocytes,macrophages and neutrophils. Immuno-histochemical methods may also beused to determine the presence of inflammatory cytokines includinginterleulin-1, TNF-alpha, and TGF-beta.

In various embodiments, tissue scaffolds of the present disclosure canbe used to treat any of a wide range of disorders. Tissue defects canarise from diverse medical conditions, including, for example,congenital malformations, traumatic injuries, infections, and oncologicresections. Thus, the tissue scaffolds can be used to repair defects inany soft tissue, e.g., tissues that connect, support, or surround otherstructures and organs of the body. The tissue scaffolds can also be usedto treat bone defects, e.g., as an articular graft to support cartilageregeneration. Soft tissue can be any non-osseous tissue.

The tissue scaffolds can be used to treat soft tissues in many differentorgan systems. These organ systems can include, but are not limited to,the muscular system, the genitourinary system, the gastroenterologicalsystem, the integumentary system, the circulatory system, and therespiratory system. The tissue scaffolds are also useful to treatconnective tissue, including the fascia, a specialized layer thatsurrounds muscles, bones and joints, of the chest and abdominal wall andfor repair and reinforcement of tissue weaknesses in urological,gynecological and gastroenterological anatomy.

In another embodiment, the tissue or organ defect is selected from thegroup consisting of skin, bone, cartilage, meniscus, dermis, myocardium,periosteum, artery, vein, stomach, small intestine, large intestine,diaphragm, tendon, ligament, neural tissue, striated muscle, smoothmuscle, bladder, urethra, ureter, and gingival.

For example, FIG. 1 illustrates implantation of a tissue scaffold in acartilage defect, according to certain embodiments. As shown, a tissuescaffold 180 can be used to treat a cartilage defect in a long bone 500(e.g., femur or humerus). In various embodiments, a scaffold 180 can beused to treat an articular surface or cartilage 510 of any joint. Invarious embodiments, the tissue scaffold 180 can be placed in a defector excised area of an articular surface or cartilage 510.

In some embodiments, the tissue scaffold 180 comprises an ATM and apolymer. In some embodiments, the ATM comprises tissues from twodifferent tissue sources, for example, cartilage 190 and demineralizedbone 200. In some embodiments, the tissue scaffold 180 can be used torepair other tissue or organ defects. In some embodiments, the tissuescaffold 180 can comprise dermis and cartilage. In some embodiments, thetissue scaffold comprises cartilage 190 without demineralized bone 200.In some embodiments, the tissue scaffold 180 can comprise demineralizedbone 200 without cartilage 190. In some embodiments, the tissue scaffold180 can comprise dermis.

In certain embodiments, a method of making a tissue scaffold comprisesdissolving a polymer in a solution; mixing the solution with aparticulate ATM to form a mixture; and molding and shaping the tissuescaffold to a stable three-dimensional structure by removing thesolvent.

FIG. 2 illustrates preparation of a three-dimensional tissue scaffold.The scaffolds can include an ATM (step 100). In some embodiments the ATMcan be derived from, for example, dermis, cartilage, bone, demineralizedbone, blood vessels, heart valves, fascia, or nerve connective tissue.In some embodiments, the particulate ATM comprises uniform sizeparticles. In some embodiments, the particulate ATM comprises a dermalATM. In some embodiments, the dermal ATM is a human tissue matrix. Insome embodiments, the dermal ATM is a porcine tissue matrix. In someembodiments, the particulate ATM is a cartilage tissue matrix, which maybe derived from human cartilage. In some embodiments, the cartilagetissue matrix is derived from porcine cartilage. In some embodiments,the particulate ATM comprises a bone tissue matrix. In some embodiments,the bone tissue matrix is derived from human bone. In some embodiments,the bone tissue matrix is derived from porcine bone.

The ATM can be selected to provide a variety of different biologic andmechanical properties. For example, the ATM can be selected to allowtissue ingrowth and remodeling to allow regeneration of tissue normallyfound at the site where the matrix is implanted. For example, the ATM,when implanted on or into cartilage, may be selected to allowregeneration of the cartilage without excessive fibrosis or scarformation. In addition, the ATM should not elicit an excessiveinflammatory reaction, and should ultimately be remodeled to producetissue similar to the original host tissue. In some embodiments, the ATMcomprises collagen, elastin, and vascular channels. In certainembodiments, the ATM can include ALLODERM® or Strattice™, which arehuman and porcine acellular dermal matrices respectively. Examples ofsuch materials may be found in U.S. Pat. Nos. 6,933,326 and 7,358,284.Alternatively, other suitable acellular tissue matrices can be used, asdescribed further below.

A particulate ATM can be prepared by cryomilling ATM (step 110). Theparticulate ATM can be derived from many different tissue sources. Thetissue sources can be, for example, dermis, cartilage, bone bloodvessels, heart valves, fascia, and nerve connective tissue. They aredescribed in detail below. In some embodiments, two or more differenttypes of tissues can be used to prepare particulate ATM.

In addition, the tissue scaffolds can include one or more polymericmaterials. In some embodiments, the polymeric materials can be selectedfrom a number of polymers. In certain embodiments, the polymers can beselected, for example, from chitosan, benzyl ester of hyaluronic acid(BHA), polycaprolactone (PCL), or poly-4-hydroxybutyrate (P4HB). In someembodiments, the polymer can be dissolved in a suitable solvent selectedfrom a number of solvents (step 120). In some embodiments, the solventcan be selected, for example, from dioxane, N-methyl-2-pyrrolidone(NMP), DMSO, or acetic acid. In one embodiment, the PCL is dissolved indioxane. In another embodiment, the PCL in dioxane or NMP solvent usedin the preparation of tissue scaffold can be about 5-30% (w/v). In afurther embodiment, the PCL in dioxane or NMP solvent used in thepreparation of tissue scaffold can be 5%, 8%, 10%, 12%, 15%, 18%, 20%,25%, or 30% (w/v), 5% to 30% (w/v), 10% to 30% (w/v) and any values inbetween. In another embodiment, the P4HB is dissolved in dioxane or NMP.In yet another embodiment, the P4HB in dioxane or NMP solvent used inthe preparation of tissue scaffold can be about 5-40% (w/v). In afurther embodiment, the P4HB in dioxane or NMP solvent used in thepreparation of tissue scaffold can be 5%, 8%, 10%, 12%, 15%, 18%, 20%,25%, 30%, or 40% (w/v), or 5% to 40% (w/v), 10% to 30% (w/v) and anyvalues in between. In another embodiment, the BHA is dissolved in DMSO.In yet another embodiment, the BHA in DMSO used in the preparation oftissue scaffold can be about 5-50% (w/v). In a further embodiment, theBHA in DMSO used in the preparation of tissue scaffold can be 5%, 8%,10%, 12%, 15%, 18%, 20%, 25%, 30%, 40%, or 50% (w/v), 5% to 50% (w/v),or 10% to 40% (w/v), and any values in between. In yet anotherembodiment, the chitosan is dissolved in acetic acid. In a furtherembodiment, the acetic acid concentration is 0.1-0.5 M (pH range2.53-2.88). In a further embodiment, the pH of the chitosan and aceticacid mixture can be 4.0-5.5. In yet another embodiment, the chitosan inacetic acid used in the preparation of tissue scaffold can be 1%, 2%,3%, 4%, or 5% (w/v), and any values in between. Each of these scaffoldmaterials may impart different properties upon the final productallowing for the manipulations of in vivo turnover/persistence,biomechanical properties, and overall biological response.

The polymer solution can be mixed with the particulate ATM (step 130).The ATM and polymer/solvent mixtures can be placed or packed into molds(step 140). In some embodiments, molds can be selected from a number ofmolds. In some embodiments, the molds can be selected from eppendorftube, metal tube, injection tube, or a mold in the form of a tissue ororgan defect for which the tissue scaffold is designed to repair.

The solvent can be removed through a drying process (step 150). In someembodiments, the solvent can be removing through a number of dryingprocesses. In some embodiments, the drying processes can be selectedfrom freeze drying, air drying, heated drying, drying in an argon ornitrogen atmosphere, or vacuum assisted drying. The resulting tissuescaffolds consist of regenerative tissue particles encased in apolymeric/synthetic support scaffold and are stable under mechanicalstress. In addition, the drying process can include passive drying,wherein the solvent evaporates into a normal atmosphere.

In certain embodiments, shape and stability of the tissue scaffold areimportant. In some embodiments, the desired or performed shape and sizeof the resulting tissue scaffold is determined by the shape and size ofa mold used to produce the scaffold. In some embodiments, the desired orperformed shape of the tissue scaffold is a stable three-dimensionalshape. In some embodiments, the mold used to prepare the stablethree-dimensional tissue scaffold can be an eppendorf tube, a metaltube, an injection tube, or a mold in the form of a tissue or organdefect in which the tissue scaffold will be implanted. In someembodiments, the tissue scaffold is in a cylindrical shape. In someembodiments, the tissue scaffold is in a tubular shape. In someembodiments, the shape of the tissue scaffold corresponds to the shapeof the tissue or organ defect or injury. In some embodiments, mechanicalstrength, porosity, hydration and fluid conductance are controlled bycontrolling freezing rate, freezing temperature, and the composition ofthe molding container.

In some embodiments, the tissue scaffold is sized or shaped such that itcan correspond to the shape of the tissue or organ defect. In someembodiments, the tissue scaffold can be prepared using two or moredifferent types of tissues. For example, one of the tissues in thetissue scaffold can be cartilage and the second tissue can bedemineralized bone. In some embodiments, cartilage/demineralized bonescaffold can be used to repair osteochondral defects (FIG. 1).

Acellular Tissue Matrices

The term “acellular tissue matrix,” as used herein, refers generally toany tissue matrix that is substantially free of cells and/or cellularcomponents. Skin, parts of skin (e.g., dermis), and other tissues suchas blood vessels, heart valves, fascia, cartilage, bone, and nerveconnective tissue may be used to create acellular matrices within thescope of the present disclosure. Acellular tissue matrices can be testedor evaluated to determine if they are substantially free of cell and/orcellular components in a number of ways. For example, processed tissuescan be inspected with light microscopy to determine if cells (live ordead) and/or cellular components remain. In addition, certain assays canbe used to identify the presence of cells or cellular components. Forexample, DNA or other nucleic acid assays can be used to quantifyremaining nuclear materials within the tissue matrices. Generally, theabsence of remaining DNA or other nucleic acids will be indicative ofcomplete decellularization (i.e., removal of cells and/or cellularcomponents). Finally, other assays that identify cell-specificcomponents (e.g., surface antigens) can be used to determine if thetissue matrices are acellular. Skin, parts of skin (e.g., dermis), andother tissues such as blood vessels, heart valves, fascia, cartilage,bone, and nerve connective tissue may be used to create acellularmatrices within the scope of the present disclosure.

In general, the steps involved in the production of an ATM includeharvesting the tissue from a donor (e.g., a human cadaver or animalsource) and cell removal under conditions that preserve biological andstructural function. For example, desired biologic and structuralfunctions include the ability to support cell ingrowth and tissueregeneration, to provide mechanical support (e.g., to a surgical site ordefect), to prevent excessive immunologic response, inflammation,fibrosis, and/or scarring. In certain embodiments, the process includeschemical treatment to stabilize the tissue and avoid biochemical andstructural degradation together with or before cell removal. In variousembodiments, the stabilizing solution arrests and prevents osmotic,hypoxic, autolytic, and proteolytic degradation, protects againstmicrobial contamination, and reduces mechanical damage that can occurwith tissues that contain, for example, smooth muscle components (e.g.,blood vessels). The stabilizing solution may contain an appropriatebuffer, one or more antioxidants, one or more oncotic agents, one ormore antibiotics, one or more protease inhibitors, and/or one or moresmooth muscle relaxants.

The tissue is then placed in a decellularization solution to removeviable cells (e.g., epithelial cells, endothelial cells, smooth musclecells, and fibroblasts) from the structural matrix without damaging thebiological and structural integrity of the collagen matrix. Theintegrity of the collagen matrix can be tested in a number of ways. Forexample, differential scanning calorimetry can be used to identifychanges in thermal transition temperature that indicate cross-linking(elevation in transition temperature) or collagen degredation (decreasein transition temperatures). In addition, electron microscopy candemonstrate changes in normal collagen patterns, and enzymatic digestionassays can demonstrate collagen damage. Further, the loss of variousglycosaminoglycans (e.g., chondroitin sulfate and hyaluronic acid) canindicate an undesirable change in the tissue matrix.

The decellularization solution may contain an appropriate buffer, salt,an antibiotic, one or more detergents (e.g., TRITON X-100™, sodiumdeoxycholate, polyoxyethylene (20) sorbitan mono-oleate), one or moreagents to prevent cross-linking, one or more protease inhibitors, and/orone or more enzymes. Suitable methods for producing ATM are described,for example, H. Xu et al., A Porcine-Derived Acellular Dermal ScaffoldThat Supports Soft Tissue Regeneration: Removal of TerminalGalactose-α-(1,3)-Galactose and Retention of Matrix Structure. TissueEng. Part A 15: 1807 (2009).

After the decellularization process, the tissue sample is washedthoroughly with saline. In some exemplary embodiments, e.g., whenxenogenic material is used, the decellularized tissue is then treatedovernight at room temperature with a deoxyribonuclease (DNase) solution.In some embodiments, the tissue sample is treated with a DNase solutionprepared in DNase buffer (20 mM HEPES(4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid), 20 mM CaCl₂ and 20mM MgCl₂). Optionally, an antibiotic solution (e.g., Gentamicin) may beadded to the DNase solution. Any suitable buffer can be used as long asthe buffer provides suitable DNase activity.

While an ATM may be made from one or more individuals of the samespecies as the recipient of the tissue scaffold, this is not necessarilythe case. Thus, for example, an ATM in the tissue scaffold may be madefrom porcine tissue. Species that can serve as recipients of ATM anddonors of tissues or organs for the production of the ATM include,without limitation, mammals, such as humans, nonhuman primates (e.g.,monkeys, baboons, or chimpanzees), pigs, cows, horses, goats, sheep,dogs, cats, rabbits, guinea pigs, gerbils, hamsters, rats, or mice.

Elimination of the α-gal epitopes from the collagen-containing materialmay diminish the immune response against the collagen-containingmaterial. The α-gal epitope is expressed in non-primate mammals and inNew World monkeys (monkeys of South America) on macromolecules such asglycoproteins of the extracellular components. U. Galili et al., J.Biol. Chem. 263: 17755 (1988). This epitope is absent in Old Worldprimates (monkeys of Asia and Africa and apes) and humans, however.Anti-gal antibodies are produced in humans and primates as a result ofan immune response to α-gal epitope carbohydrate structures ongastrointestinal bacteria. U. Galili et al., Infect. Immun. 56: 1730(1988); R. M. Hamadeh et al., J. Clin. Invest. 89: 1223 (1992).

Since non-primate mammals (e.g., pigs) produce α-gal epitopes,xenotransplantation of collagen-containing material from these mammalsinto primates often results in rejection because of primate anti-Galbinding to these epitopes on the collagen-containing material. Thebinding results in the destruction of the collagen-containing materialby complement fixation and by antibody dependent cell cytotoxicity. U.Galili et al., Immunology Today 14: 480 (1993); M. Sandrin et al., Proc.Natl. Acad. Sci. USA 90: 11391 (1993); H. Good et al., Transplant. Proc.24: 559 (1992); B. H. Collins et al., J. Immunol. 154: 5500 (1995).Furthermore, xenotransplantation results in major activation of theimmune system to produce increased amounts of high affinity anti-galantibodies. Accordingly, in some embodiments, when animals that produceα-gal epitopes are used as the tissue source, the substantialelimination of α-gal epitopes from cells and from extracellularcomponents of the collagen-containing material, and the prevention ofre-expression of cellular α-gal epitopes can diminish the immuneresponse against the collagen-containing material associated withanti-gal antibody binding to α-gal epitopes.

To remove α-gal epitopes, after washing the tissue thoroughly withsaline to remove the DNase solution, the tissue sample may be subjectedto one or more enzymatic treatments to remove certain immunogenicantigens, if present in the sample. In some embodiments, the tissuesample may be treated with an α-galactosidase enzyme to eliminate α-galepitopes if present in the tissue. In some embodiments, the tissuesample is treated with α-galactosidase at a concentration of 300 U/Lprepared in 100 mM phosphate buffer at pH 6.0. In other embodiments, theconcentration of α-galactosidase is increased to 400 U/L for adequateremoval of the α-gal epitopes from the harvested tissue. Any suitableenzyme concentration and buffer can be used as long as sufficientremoval of antigens is achieved.

Alternatively, rather than treating the tissue with enzymes, animalsthat have been genetically modified to lack one or more antigenicepitopes may be selected as the tissue source. For example, animals(e.g., pigs) that have been genetically engineered to lack the terminalα-galactose moiety can be selected as the tissue source. Fordescriptions of appropriate animals see co-pending U.S. application Ser.No. 10/896,594 and U.S. Pat. No. 6,166,288, the disclosures of which areincorporated herein by reference in their entirety. In addition, certainexemplary methods of processing tissues to produce acellular matriceswith or without reduced amounts of or lacking alpha-1,3-galactosemoieties, are described in Xu, Hui. et al., “A Porcine-Derived AcellularDermal Scaffold that Supports Soft Tissue Regeneration: Removal ofTerminal Galactose-α-(1,3)-Galactose and Retention of Matrix Structure,”Tissue Engineering, Vol. 15, 1-13 (2009), which is incorporated byreference in its entirety.

After the ATM is formed, histocompatible, viable cells may optionally beseeded in the ATM to produce a graft that may be further remodeled bythe host. In some embodiments, histocompatible viable cells may be addedto the matrices by standard in vitro cell co-culturing techniques priorto transplantation, or by in vivo repopulation followingtransplantation. In vivo repopulation can be by the recipient's owncells migrating into the ATM or by infusing or injecting cells obtainedfrom the recipient or histocompatible cells from another donor into theATM in situ. Various cell types can be used, including embryonic stemcells, adult stem cells (e.g. mesenchymal stem cells), and/or neuronalcells. In various embodiments, the cells can be directly applied to theinner portion of the ATM just before or after implantation. In certainembodiments, the cells can be placed within the ATM to be implanted, andcultured prior to implantation. In one embodiment, viable cells areadded to the tissue scaffold prior to implantation. In one embodiment,viable cells are added to the tissue scaffold after the scaffold isimplanted at a desired anatomic site.

Particulate Acellular Tissue Matrix

The following procedure can be used to produce particulate acellulartissue matrices using ALLODERM®, STRATTICE™ LifeCell Corporation,Branchburg, N.J., or other suitable acellular tissue matrices. Afterremoval from the packaging, ATM is cut into strips using a Zimmer mesherfitted with a non-interrupting “continuous” cutting wheel. The resultinglong strips of ATM are cut into lengths of about 1 to about 2centimeters in length.

A homogenizer and sterilized homogenizer probe, such as a LabTeck Macrohomogenizer available from OMNI International, Warrenton Va., isassembled and cooled to cryogenic temperatures using sterile liquidnitrogen which is poured into the homogenizer tower. Once thehomogenizer has reached cryogenic temperatures, ATM previously preparedinto strips as noted above are added to the homogenizing towercontaining sterile liquid nitrogen. The homogenizer is then activated soas to cryogenically fracture the strips of ATM. The time and duration ofthe cryogenic fractionation step will depend upon the homogenizerutilized, the size of the homogenizing chamber, the speed and time atwhich the homogenizer is operated and should be able to be determined byone of skill in the art by simple variation of the parameters to achievethe desired results.

The cryofractured particulate ATM material is sorted by particle size bywashing the product of the homogenizer with liquid nitrogen through aseries of metal screens that have also been cooled to liquid nitrogentemperatures. We have found it especially useful to utilize acombination of screens within the homogenizing tower of the typedescribed above in which the particles are washed and sorted first toexclude oversized particles and then to exclude undersized particles.

Once isolated, the particulate ATM is removed and placed in a vial forfreeze drying once the sterile liquid nitrogen has evaporated. This laststep is to ensure that any residual moisture that may have been absorbedduring the above procedure is removed.

The final product can be a powder having a particle size of about 1micron to about 900 microns or a particle size of about 30 microns toabout 750 microns. The particles are distributed about a mean of about150-300 microns. The material is readily rehydrated by suspension innormal saline or other similar suitable rehydrating agent. Therehydrated ATM may be resuspended in normal saline or any other suitablepharmaceutically compatible carrier.

The following examples are provided to better explain the variousembodiments and should not be interpreted in any way to limit the scopeof the present disclosure.

EXAMPLES Example 1 Preparation of Regenerative Tissue Scaffold

FIG. 2 illustrates a process of preparation of synthetic ATMregenerative tissue scaffold. Three dimensional regenerative tissuescaffolds were created from particulate ATM using polymeric scaffoldmaterials. ATM was prepared from porcine dermal tissue and freeze dried.Dry ATM was cut into ˜1 cm² pieces and placed into an appropriate sizecryomill vial. The vial was then placed in a SPEX 6800 freezer mill thathas been pre-cooled with liquid nitrogen and subjected to a cryofractureprotocol. The particulate ATM was then removed from the vial andmaintained under dry storage conditions.

A 100% benzyl ester derivative of hyaluronic acid was solubilized indimethyl sulfoxide (DMSO) at a concentration of 40% (w/v). One ml ofthis solution was then mixed with 300 mg of particulate acellular dermalmatrix, as prepared above, and the mixture was transferred to a small 2ml eppendorf tube. The DMSO was then removed through a freeze dryprocess leaving a tissue scaffold (40% benzyl ester hyaluronic acid, 60%acellular dermal matrix by weight) that retained the form of theeppendorf tube (cylindrical) it was dried in (step 150).

Example 2 Functional Study of Tissue Scaffold

Calorimetric Analysis of Effect of Organic Solvents on Tissue MatrixIntegrity

In vivo results suggested that the presence of the regenerative tissuematrix attenuates the immunological or inflammatory response to thescaffold material as demonstrated by reduced number of inflammatorycells.

pADMs, prepared as described in Example 1, were treated with an excessof different solvents including dioxane, NMP, and DMSO, for 2 hr. Thetreated materials were evaluated with differential scanning calorimeter(DSC) to assess tissue matrix integrity. FIG. 3 is a graph of DSC dataof porcine acellular dermal matrix (pADM) treated with organic solvents.

Similarly, pADMs were treated with different polymers, for example,poly-4-hydroxybutyrate in dioxane or NMP and polycaprolactone in dioxaneor NMP, and evaluated tissue matrix integrity by DSC. FIG. 4 is a graphof DSC data of pADM in presence of polymers, according to Example 2. DSCanalysis (FIG. 4) showed that thermograms of polymer and pADM wereadditive.

Histological Evaluation

The effect of implantation of pADM was tested in the presence ofpoly-4-hydroxybutyrate (P4HB) in a sub-dermal immune-competent ratmodel. Immune-competent rats were implanted with pADM/P4HB tissuescaffold or P4HB polymer scaffold. This model allowed for thedetermination of cellular and immunological responses to the implantedtest materials. Test materials were implanted in a sub-dermal positionthrough a small incision on the dorsal surface of immune-competent rats(Rattus norvegicus; Lewis Rat). Four weeks (FIGS. 5A-D) and 12 weeks(FIGS. 6A-D) after implantation, explants were collected and washed withPBS and were fixated in 10% formalin. Fixed tissue was embedded inparaffin and sections of tissue matrix samples were stained withhematoxylin and eosin (H&E) using standard procedures. D. C. Sheehan andB. B. Hrapchak, Theory and Practice of Histotechnology, 2^(nd) edn.,Columbus, Ohio, Battelle Press (1987). Samples were then observed undermicroscope at 100× magnification (FIGS. 5A-B and 6A-B) and 400×magnification (FIGS. 5C-D and 6C-D). Histology analysis of the explants(FIGS. 5A-D and 6A-D) showed that P4HB in the presence of pADM had anattenuated inflammatory response compared to explants of P4HB alone.

FIGS. 7A-D and 8A-D show histological evaluation of 4 and 12 weekpolycaprolactone explants. The effect of implantation of pADM was testedin the presence of polycaprolactone (PCL) in a sub-dermalimmune-competent rat model. Immune-competent rats were implanted withpADM/PCL scaffold or PCL polymer scaffold. Four (FIGS. 7A-D) and 12weeks (FIGS. 8A-D) after implantation, explants were collected andprocessed for histological evaluation, as described above. Samples werethen observed under microscope at 100× magnification (FIGS. 7A-B and8A-B) and 400× magnification (FIGS. 7C-D and 8C-D). Histology analysisof the explants (FIGS. 7A-D and 8A-D) showed that PCL in the presence ofpADM had an attenuated inflammatory response compared to explants of PCLalone.

FIGS. 9A-D and 10A-D show histological evaluation of 4 and 12 weekhyaluronic acid benzyl ester (BHA) explants. The effect of implantationof pADM was tested in the presence of BHA in a sub-dermalimmune-competent rat model. Immune-competent rats were implanted withpADM/BHA scaffold or BHA polymer scaffold. Four (FIGS. 9A-D) and 12weeks (FIGS. 10A-D) after implantation, explants were collected andprocessed for histological evaluation, as described above. Samples werethen observed under microscope at 100× magnification (FIGS. 9A-B and10A-B) and 400× magnification (FIGS. 9C-D and 10C-D). Histology analysisof the explants (FIGS. 9A-D and 10A-D) showed that BHA in the presenceof pADM had an attenuated inflammatory response compared to explants ofBHA alone.

FIGS. 11A-D show histological evaluation of 4 and 8 week chitosan/pADMexplants. The effect of implantation of pADM was tested in the presenceof chitosan in a sub-dermal immune-competent rat model. Immune-competentrats were implanted with pADM/chitosan scaffold. Four (FIGS. 11A, C) and8 weeks (FIGS. 11B, D) after implantation, explants were collected andprocessed for histological evaluation, as described above. Samples werethen observed under microscope at 100× magnification (FIGS. 11A-B) and400× magnification (FIGS. 11C-D). The results showed that there was aregenerative tissue response, as demonstrated by the presence offibroblast-like cells and blood vessels.

The capacity to mold and shape regenerative tissue matrices into stablethree-dimensional structures, as described above, will allow for thedevelopment of novel products that can be used over a broad range ofregenerative medical applications. Each of these scaffold materials mayimpart different properties upon the final product allowing for themanipulations of in vivo turnover/persistence, biomechanical propertiesand overall biological response. In addition, as shown above, theregenerative tissue matrix component reduces/attenuates the overallinflammatory response to the scaffold materials.

1. A method for making a tissue scaffold, comprising: dissolving apolymer in a solvent to make a solution; mixing the solution with aparticulate acellular tissue matrix (ATM) to create a mixture; placingthe mixture in a mold; and drying the mixture to form a tissue scaffoldwith a stable three-dimensional shape, wherein the tissue scaffold has areduced immunological or inflammatory response when implanted in a humanthan the polymer alone.
 2. The method of claim 1, wherein the polymercomprises a polycaprolactone.
 3. The method of claim 2, wherein thesolvent comprises dioxane.
 4. The method of claim 2, wherein the solventcomprises N-methyl-2-pyrrolidone.
 5. The method of claim 2, wherein thepolycaprolactone in solvent is present in an amount ranging from about5-30% (w/v).
 6. The method of claim 2, wherein the polycaprolactone insolvent is present in an amount ranging from about 10-30% (w/v).
 7. Themethod of claim 1, wherein the polymer is a poly-4-hydroxybutyrate. 8.The method of claim 7, wherein the solvent comprises dioxane.
 9. Themethod of claim 7, wherein the solvent comprises N-methyl-2-pyrrolidone.10. The method of claim 7, wherein the poly-4-hydroxybutyrate in solventis present in an amount ranging from about 5-40% (w/v).
 11. The methodof claim 7, wherein the poly-4-hydroxybutyrate in solvent is present inan amount ranging from about 10-30% (w/v).
 12. The method of claim 1,wherein the polymer comprises a benzyl ester derivative of hyaluronicacid.
 13. The method of claim 12, wherein the solvent comprises DMSO.14. The method of claim 12, wherein the benzyl ester derivative ofhyaluronic acid in solvent is present in an amount ranging from about5-50% (w/v).
 15. The method of claim 12, wherein the benzyl esterderivative of hyaluronic acid in solvent is present in an amount rangingfrom about 10-40% (w/v).
 16. The method of claim 1, wherein the polymercomprises chitosan.
 17. The method of claim 16, wherein the solventcomprises acetic acid.
 18. The method of claim 17, wherein the aceticacid is 0.1-0.5 M.
 19. The method of claim 16, wherein the chitosan ispresent in an amount ranging from about 1-5% (w/v).
 20. The method ofclaim 1, wherein the particulate ATM comprises uniform size particles.21. The method of claim 1, wherein the particulate ATM comprises adermal ATM.
 22. The method of claim 21, wherein the dermal ATM is ahuman tissue matrix.
 23. The method of claim 21, wherein the dermal ATMis a porcine tissue matrix.
 24. The method of claim 1, wherein theparticulate ATM is a cartilage tissue matrix.
 25. The method of claim24, wherein the cartilage tissue matrix comprises a human cartilagematrix.
 26. The method of claim 24, wherein the cartilage tissue matrixcomprises a porcine cartilage matrix.
 27. The method of claim 1, whereinthe particulate ATM comprises a bone tissue matrix.
 28. The method ofclaim 27, wherein the bone tissue matrix comprises a human bone.
 29. Themethod of claim 27, wherein the bone tissue matrix comprises a porcinebone.
 30. The method of claim 1, wherein placing the mixture in a moldcomprises injection molding.
 31. The method of claim 1, wherein theparticulate ATM comprises ATM from two or more different types oftissues.
 32. The method of claim 31, wherein the two or more differenttypes of tissues comprise dermis and cartilage.
 33. The method of claim31, wherein the two or more different types of tissues comprisecartilage and bone.
 34. The method of claim 31, wherein the two or moredifferent types of tissues comprise human tissue matrices.
 35. Themethod of claim 31, wherein the two or more different types of tissuescomprise porcine tissue matrices.
 36. The method of claim 31, whereinthe two or more different types of tissues comprise human tissue matrixand porcine tissue matrix.
 37. A tissue scaffold, comprising: aparticulate acellular tissue matrix (ATM); and a polymer; wherein theATM is encased in the polymer to form a stable three-dimensional tissuescaffold for tissue regeneration, and wherein the tissue scaffold has areduced immunological or inflammatory response when implanted in a humanthan the polymer alone.
 38. The tissue scaffold of claim 37, wherein thepolymer comprises a synthetic polymer.
 39. The tissue scaffold of claim37, wherein the polymer comprises a polycaprolactone.
 40. The tissuescaffold of claim 37, wherein the polymer is a poly-4-hydroxybutyrate.41. The tissue scaffold of claim 37, wherein the polymer comprises abenzyl ester derivative of hyaluronic acid.
 42. The tissue scaffold ofclaim 37, wherein the polymer comprises chitosan.
 43. The tissuescaffold of claim 37, wherein the particulate ATM comprises a dermalATM.
 44. The tissue scaffold of claim 43, wherein the dermal ATM is ahuman tissue matrix.
 45. The tissue scaffold of claim 43, wherein thedermal ATM is a porcine tissue matrix.
 46. The tissue scaffold of claim37, wherein the ATM is a cartilage tissue matrix.
 47. The tissuescaffold of claim 46, wherein the cartilage tissue matrix comprises ahuman cartilage.
 48. The tissue scaffold of claim 46, wherein thecartilage tissue matrix comprises a porcine cartilage.
 49. The tissuescaffold of claim 37, wherein the ATM is a bone tissue matrix.
 50. Thetissue scaffold of claim 49, wherein the bone tissue matrix comprises ahuman bone.
 51. The tissue scaffold of claim 49, wherein the bone tissuematrix comprises a porcine bone.
 52. A method of treatment, comprising:(a) selecting a tissue scaffold having a stable three-dimensional shape,the tissue scaffold comprising: (i) a particulate acellular tissuematrix (ATM); and (ii) a polymer; wherein the ATM is encased in thepolymer to form a stable three-dimensional tissue scaffold for tissueregeneration, and wherein the tissue scaffold has a reducedimmunological or inflammatory response when implanted in a human thanthe polymer alone; (b) identifying a defect in a tissue or organ; and(c) implanting the tissue scaffold in the defect.
 53. The method ofclaim 52, wherein the defect is in a bone.
 54. The method of claim 52,wherein the defect is in a cartilaginous tissue.
 55. The method of claim52, wherein the defect is in a breast tissue.
 56. A regenerative tissuescaffold made according to the method of claim 1.